Efficient method for displaying protein multimer

ABSTRACT

The invention provides a method of producing a protein multimer-nucleic acid complex comprising a protein multimer and any one target component of the protein multimer. A nucleic acid encoding the target component is subjected to in vitro translation to provide a translation product containing a target component-nucleic acid complex of the target component and the nucleic acid encoding the target component. A nucleic acid encoding a non-target component constituting the protein multimer together with the target component is translated into the non-target component by adding the nucleic acid encoding the non-target component to the previously provided translation product to provide the non-target component. The non-target component then is associated with the target component contained in the target component-nucleic acid complex to form a protein multimer, thus affording the protein multimer-nucleic acid complex of the protein multimer and the nucleic acid encoding the target component.

CROSS-REFERENCE TO RELATED APPLICATIONS

This patent application is a continuation of U.S. patent applicationSer. No. 13/871,530, filed Apr. 26, 2013, which issued as U.S. Pat. No.9,289,742, which claims the benefit of U.S. Provisional PatentApplication No. 61/638,774 filed on Apr. 26, 2012, which is incorporatedby reference in its entirety herein.

INCORPORATION-BY-REFERENCE OF MATERIAL ELECTRONICALLY SUBMITTED

Incorporated by reference in its entirety herein is a computer-readablenucleotide/amino acid sequence listing submitted concurrently herewithand identified as follows: 4,333 bytes ASCII (Text) file named“723263SequenceListing.txt,” created Feb. 12, 2016.

TECHNICAL FIELD OF THE INVENTION

The present invention relates to a method of efficiently preparing acomplex containing a protein multimer and a nucleic acid in an in vitrotranslation system, which is used for selecting, from the gene library,a nucleic acid encoding a component contained in a protein multimerhaving a desired function.

BACKGROUND OF THE INVENTION

Monoclonal antibody which has been vastly developed as an antibodypharmaceutical product in recent years is a protein multimer consistingof H chain and L chain. Many improving techniques have been proposed inan attempt to enhance its drug efficacy and the like. For example, ithas been shown that shuffling of the genes of H chain and L chain of anantibody is effective for improving the affinity of the antibody. Thatis, an antibody protein (e.g., Fab-type antibody) which is translatedfrom the gene of either H chain library or L chain library and thecorresponding partner chain are displayed in an in vivo selection systemsuch as phage display (non-patent document 1: J. Mol. Biol.,1996.255.28-43, non-patent document 2: Biochem. Biophys. Res. Commun.,2002.295.31-36), yeast surface display (non-patent document 3: ProteinEng. Des. Sel., 2010.23.311-319) and the like, and selections areperformed based on the affinity for the target antigen, whereby a clonewith a higher affinity can be selected. In phage display and yeastsurface display, however, a preparation of a library having diversityexceeding 10¹⁰ molecules is so hard that it requires substantial effortsand time, since the insertion efficiency of gene into an expressionvector and the transformation efficiency of host cells are limited(generally 10⁷-10¹⁰ molecules).

On the other hand, an in vitro selection system utilizing an in vitrotranslation system, which is represented by ribosome display (patentdocument 1: JP-A-2008-271903), is a technique for forming a complex of agene (mRNA or cDNA) and a protein encoded by the gene in the in vitrotranslation system, and efficiently selecting a protein having aparticular function. The essential element here is that the genotype andthe phenotype correspond one-to-one. Specifically, cDNA or mRNA as agenotype needs to be physically bound one-to-one with a proteintranslated therefrom. Since a complex having a desired function isselected based on the properties of a protein phenotype, the number ofthe complexes shows the diversity of the library. Preparation of acomplex in an in vitro selection system does not require a step oftransformation of living cells, which is necessary for the in vivoselection system such as yeast surface display and the like, and can beachieved by the addition of a gene library to the in vitro translationsystem. Therefore, a library of complexes exceeding 10¹³ molecules canbe easily prepared in the in vitro selection system, and a desiredfunctional molecule can be selected using the library.

As mentioned above, the in vitro selection system is constituted byforming a one-to-one complex of a gene (mRNA or DNA) and a proteinencoded by the gene by adding a gene library to an in vitro translationsystem. Therefore, if the protein to be selected is a protein multimerconsisting of plural gene products, it is difficult to prepare agene-protein complex.

SUMMARY OF THE INVENTION

When the affinity of an antibody, which is a representative proteinmultimer, is to be improved in an in vitro selection system, forexample, L chain library is displayed on the ribosome and H chain isallowed to exist as a free protein in the same system to form anantibody, which is a heterodimer of L chain and H chain, and a complexcontaining antibody, ribosome and mRNA (i.e., ribosome display complex)needs to be formed finally in the system. In the ribosome display,ribosome, which is translating in vitro, needs to stably stay on themRNA, and therefore, treatments such as removal of stop codon from Lchain mRNA, addition of a sequence encoding translation elongationarrest sequence (arrest sequence) to the 3′-side of L chain mRNA,removal of a release factor or ribosome recycling factor from the invitro translation system and the like are generally performed (patentdocument 1: JP-A-2008-271903). Meanwhile, since H chain needs to betranslated as a free protein, treatments to keep ribosome on mRNA ratherdecrease the translation efficiency. Therefore, when mRNAs of H chainand L chain requiring different translation regulation aresimultaneously translated in vitro within the same system, both thedisplay efficiency of L chain on ribosome and the translation efficiencyof H chain decrease, which in turn markedly decreases the formationefficiency of the ribosome display complex.

Accordingly, there is a demand for the development of a method ofefficiently displaying a protein multimer such as antibody in an invitro selection system capable of preparing libraries with highdiversity, for example, ribosome display.

An object of the present invention is to provide a means for, in an invitro selection system, more efficiently displaying a protein multimer,efficiently enhancing or improving functions (e.g., affinity and thelike) of a protein multimer such as antibody and the like, or selectinga nucleic acid encoding a component contained in a protein multimerhaving a desired function.

The present inventors have conducted intensive studies in an attempt tosolve the above-mentioned problems and found that the mRNA recovery rateafter selection is drastically improved, by first forming a ribosomedisplay complex containing a library gene (L chain of antibody here),and then expressing the pair protein gene (H chain of antibody here) inthe same tube, or by performing formation of a ribosome display complexcontaining a library gene (L chain of antibody here) and synthesis ofthe pair protein gene (H chain of antibody here) in separate tubes, andmixing the reaction products in those tubes, compared to the case whereL chain and H chain are simultaneously translated in the same tube.

More specifically, as a first step of the first embodiment, a spacergene for ribosome display (geneIII, tolA and the like) was ligated tothe 3′ side of the gene of L chain. Furthermore, a stop codon wasremoved from the spacer sequence, or the arrest sequence of Escherichiacoli was added to the 3′-terminus of the spacer gene, and the obtainedmRNA was added to an in vitro translation system, whereby a Lchain-ribosome-mRNA of L chain ternary complex (ribosome displaycomplex) was formed. As a second step, mRNA of H chain and ribosomeprepared in advance were added to the reaction mixture containing theribosome display complex to synthesize a free H chain, which was thenassociated with the L chain displayed on the ribosome display complex,whereby a ribosome display complex displaying the antibody consisting ofH chain and L chain was finally formed with high efficiency.

In a second embodiment, the same reaction as in the first step of theabove-mentioned first embodiment was performed in one tube, then H chainwas synthesized by subjecting mRNA of H chain (or coding for H chain) toan in vitro translation system in a separate tube. When the reactionproducts in the two tubes were mixed, H chain was associated with the Lchain displayed on the ribosome display complex, whereby a ribosomedisplay complex displaying the antibody consisting of H chain and Lchain was finally formed with high efficiency.

They have further studied based on these findings and completed thepresent invention. Accordingly, the present invention relates to thefollowing.

[1] A method of producing a protein multimer-nucleic acid complexcomprising the protein multimer and a nucleic acid encoding any onetarget component of the protein multimer, which comprises the followingsteps:

i) translating a nucleic acid encoding the target component into thetarget component by an in vitro translation system to give a translationproduct containing a target component-nucleic acid complex containingthe target component and the nucleic acid encoding the target component,andii) translating a nucleic acid encoding a non-target componentconstituting the protein multimer together with the target componentinto the non-target component by adding the nucleic acid encoding thenon-target component to the translation product obtained in i) to givethe non-target component, associating the non-target component with thetarget component in the target component-nucleic acid complex to formthe protein multimer, thus affording the protein multimer-nucleic acidcomplex comprising the protein multimer and the nucleic acid encodingthe target component.[2] The method of [1], wherein the in vitro translation system in i)does not comprise a nucleic acid encoding the non-target component.[3] The method of [1] or [2], wherein the protein multimer-nucleic acidcomplex and the target component-nucleic acid complex comprise aribosome.[4] The method of any of [1]-[3], wherein ii) comprises adding aribosome to the translation product obtained in i).[5] The method of any of [1]-[4], wherein one molecule of the proteinmultimer and one molecule of the nucleic acid encoding the targetcomponent are contained per one molecule of the protein multimer-nucleicacid complex.[6] The method of any of [1]-[5], wherein the in vitro translationsystem consists of independently purified factors.[7] The method of [6], wherein at least one of the independentlypurified factors is a factor extracted from prokaryote.[8] The method of any of [1]-[7], wherein the protein multimer is adimer.[9] The method of any of [1]-[8], wherein the protein multimer has afunction as a multimer.[10] The method of any of [1]-[9], wherein the protein multimer is anantibody.[11] The method of [10], wherein the antibody is a Fab fragment.[12] The method of [10] or [11], wherein the target component is oneselected from the group consisting of an L chain and an H chain, and thenon-target component is the other.[13] The method of any of [1]-[12], wherein the nucleic acid encodingthe target component is a library of the nucleic acid encoding thetarget component.[14] A method of producing a library of a protein multimer-nucleic acidcomplex comprising the protein multimer and a nucleic acid encoding anyone target component of the protein multimer, which comprises thefollowing steps:i) translating a library of nucleic acid encoding the target componentinto the target component by an in vitro translation system to give atranslation product containing a library of a target component-nucleicacid complex containing the target component and the nucleic acidencoding the target component, andii) translating a nucleic acid encoding a non-target componentconstituting the protein multimer together with the target componentinto the non-target component by adding the nucleic acid encoding thenon-target component to the translation product obtained in i) to givethe non-target component, associating the non-target component with thetarget component in the target component-nucleic acid complex to formthe protein multimer, thus affording the library of the proteinmultimer-nucleic acid complex comprising the protein multimer and thenucleic acid encoding the target component.[15] A method of producing a protein multimer-nucleic acid complexcomprising the protein multimer and a nucleic acid encoding any onetarget component of the protein multimer, which comprises the followingsteps:i′) translating the nucleic acid encoding the target component into thetarget component by an in vitro translation system to give a translationproduct containing a target component-nucleic acid complex containingthe target component and the nucleic acid encoding the target component,ii′) translating a nucleic acid encoding a non-target componentconstituting the protein multimer together with the target component byan in vitro translation system to give a translation product containingthe non-target component, andiii′) mixing the translation product of i′) with the translation productof ii′), associating the non-target component with the target componentcontained in the target component-nucleic acid complex to form theprotein multimer, thus affording the protein multimer-nucleic acidcomplex comprising the protein multimer and the nucleic acid encodingthe target component.[16] The method of [15], wherein the in vitro translation system in i′)does not contain the nucleic acid encoding the non-target component.[17] The method of [15] or [16], wherein the in vitro translation systemin ii′) does not contain the nucleic acid encoding the target component.[18] The method of any of [15]-[17], wherein the proteinmultimer-nucleic acid complex and the target component-nucleic acidcomplex further comprise a ribosome.[19] The method of any of [15]-[18], wherein one molecule of the proteinmultimer, and one molecule of the nucleic acid encoding the targetcomponent are contained per one molecule of the protein multimer-nucleicacid complex.[20] The method of any of [15]-[19], wherein the in vitro translationsystem consists of independently purified factors.[21] The method of [20], wherein at least one of the independentlypurified factors is a factor extracted from prokaryote.[22] The method of any of [15]-[21], wherein the protein multimer is adimer.[23] The method of any of [15]-[22], wherein the protein multimer has afunction as a multimer.[24] The method of any of [15]-[23], wherein the protein multimer is anantibody.[25] The method of [24], wherein the antibody is a Fab fragment.[26] The method of [24] or [25], wherein the target component is oneselected from the group consisting of an L chain and an H chain, and thenon-target component is the other.[27] The method of any of [15]-[26], wherein the nucleic acid encodingthe target component is a library of the nucleic acid encoding thetarget component.[28] A method of producing a library of a protein multimer-nucleic acidcomplex comprising the protein multimer and a nucleic acid encoding anyone target component of the protein multimer, which comprises thefollowing steps:i′) translating a library of a nucleic acid encoding the targetcomponent into the target component by an in vitro translation system togive a translation product containing a library of a targetcomponent-nucleic acid complex containing the target component and thenucleic acid encoding the target component,ii′) translating a nucleic acid encoding a non-target componentconstituting the protein multimer together with the target component byan in vitro translation system to give a translation product containingthe non-target component, andiii′) mixing the translation product of i′) with the translation productof ii′), associating the non-target component with the target componentcontained in the target component-nucleic acid complex to form theprotein multimer, thus affording the library of the proteinmultimer-nucleic acid complex comprising the protein multimer and thenucleic acid encoding the target component.

Effect of the Invention

According to the present invention, a nucleic acid encoding a componentcontained in a protein multimer having a desired function can beselected with a high efficiency from the library, since the proteinmultimer can be efficiently displayed on a display complex. Byperforming in vitro selection such as a ribosome display, an mRNAdisplay and the like using the present invention, the function such asbinding affinity and the like of a complicated protein multimer such asantibody and the like can be increased or improved efficiently.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows comparison of the amount of mRNA recovery from the ribosomedisplay complexes formed under two different conditions (conditions 1and 2) in percent.

FIG. 2 shows comparison of the amount of mRNA recovery from the ribosomedisplay complexes formed under two different conditions (conditions 1and 3) in percent.

DESCRIPTION OF EMBODIMENTS

In the first embodiment, the present invention provides a method ofproducing a protein multimer-nucleic acid complex comprising the proteinmultimer and a nucleic acid encoding any one target component of theprotein multimer, which comprises the following steps:

i) translating the nucleic acid encoding the target component into thetarget component by an in vitro translation system to give a translationproduct containing a target component-nucleic acid complex containingthe target component and the nucleic acid encoding the target component,andii) translating a nucleic acid encoding a non-target componentconstituting the protein multimer together with the target componentinto the non-target component by adding the nucleic acid encoding thenon-target component to the translation product obtained in i) to givethe non-target component, associating the non-target component with thetarget component in the target component-nucleic acid complex to formthe protein multimer, thus affording the protein multimer-nucleic acidcomplex comprising the protein multimer and the nucleic acid encodingthe target component.

In the second embodiment, the present invention provides a method ofproducing a protein multimer-nucleic acid complex comprising the proteinmultimer and a nucleic acid encoding any one target component of theprotein multimer, which comprises the following steps:

i′) translating the nucleic acid encoding the target component into thetarget component by an in vitro translation system to give a translationproduct containing a target component-nucleic acid complex containingthe target component and the nucleic acid encoding the target component,ii′) translating a nucleic acid encoding a non-target componentconstituting the protein multimer together with the target component byan in vitro translation system to give a translation product containingthe non-target component, andiii′) mixing the translation product of i′) with the translation productof ii′), associating the non-target component with the target componentcontained in the target component-nucleic acid complex to form theprotein multimer, thus affording the protein multimer-nucleic acidcomplex comprising the protein multimer and the nucleic acid encodingthe target component.

The method of the present invention enables, in a display techniqueutilizing an in vitro translation system, selection of a nucleic acidencoding a component contained in the multimer by physically linkingone-to-one the protein multimer and the nucleic acid encoding thecomponent contained in the protein multimer, based on the activity ofthe whole protein multimer (affinity for particular substance etc.)rather than the activity of a single component.

Examples of the display technique utilizing an in vitro translationsystem include ribosome display (Proc. Natl. Acad. Sci. USA, 91,9022-9026, 1994; Proc. Natl. Acad. Sci. USA, 94, 4937-4942, 1997;JP-A-2008-271903), mRNA display (FEBS Lett., 414.405-408, 1997), CISdisplay (Proc. Natl. Acad. Sci. USA, 101, 2806-2810, 2004), Covalentantibody display (Nucleic Acids Res., 33(1), e10, 2005) and the like.The method of the present invention can be utilized in any displaytechnique as long as it contains a step of translating a nucleic acidencoding a protein of interest into the protein to prepare a complexcontaining the protein and the nucleic acid encoding the protein, andcorresponding, one-to-one, the protein and the nucleic acid encoding theprotein.

Examples of other method utilizing an in vitro translation systeminclude a STABLE method (FEBS Lett., 457, 227-230, 1999), a microbeadsdisplay method (FEBS Lett., 532, 455-458, 2002) and the like. They cancommonly correspond to a gene of a target component and the targetcomponent by transcribing and translating the gene in a reversed-phaseemulsion. Unlike the aforementioned non-encapsulated type methods(ribosome display, mRNA display, CIS display and Covalent antibodymethod), these methods can transcribe and translate a gene andcorrespond the completed target component to the gene in an encapsulatedspace of a reversed-phase emulsion, without a special gene alterationsuch as addition of the arrest sequence, deletion of a stop codon andthe like. When a target component constitutes a protein multimer,therefore, application of the present invention is not entirelynecessary. Accordingly, the method of the present invention ispreferably used when the in vitro translation system is of anon-encapsulated type.

In the present specification, the “protein multimer” means a multimerwherein the same or different, two or more proteins are linked directlyor via a linker. The linkage between the proteins may be made by acovalent bond or a non-covalent interaction. Examples of the linkerinclude a disulfide bond formed by a reaction between cysteine residuesin proteins and the like. In contrast, when a linker is not present, themultimer may be simply formed by the affinity between proteins (hydrogenbond interaction, hydrophobic bond interaction etc.).

In the present invention, the protein multimer is a hetero or homomultimer, preferably a hetero multimer. While the number of componentscontained in a protein multimer is not particularly limited, the proteinmultimer can be a dimer, a trimer, a tetramer, a hexamer and the like,preferably a dimer.

Examples of the protein multimer include extracellularly secretedproteins and proteins on a cellular membrane such as antibody, ligand,adhesion molecule, pump, channel, receptor and the like, intracellularproteins such as signaling factor, nuclear receptor, transcriptionfactor and the like. They are not particularly limited as long as theycan form a multimer in an in vitro translation system, and have aparticular function as a multimer (for example, affinity for particularsubstance). The protein multimer is preferably an antibody.

In the present specification, the “antibody” is used as one encompassinga full-length antibody and an antigen binding fragment (i.e., “antigenbinding portion”) thereof, which is a protein multimer. The “antibody”refers to a protein multimer containing at least one H chain and one Lchain or an antigen binding portion thereof, which is a proteinmultimer.

In the present specification, the “antigen binding portion” of anantibody is used as one referring to one or more fragments of anantibody retaining an ability to specifically bind to an antigen. It hasbeen clarified that the antigen binding function of an antibody isperformed by a fragment of a full-length antibody. Examples of the“antigen binding portion” of an antibody include (i) Fab fragment, amonovalent fragment constituted by V_(L), V_(H), C_(L) and C_(H1)domains, (ii) F(ab′)₂ fragment, a divalent fragment containing two Fabfragments linked by disulfide bond in the hinge region, (iii) Fab′fragment, an inherent Fab having a hinge region portion (see FUNDAMENTALIMMUNOLOGY, Paul ed., 3.sup.rd ed. 1993) and the like. The antigenbinding portion generally contains at least one H chain and at least oneL chain. The Fab fragment is a protein dimer constituted by one H chainand one L chain. The F(ab′)₂ fragment is a protein tetramer constitutedby two H chains and two L chains. The Fab′ fragment is a protein dimerconstituted by one H chain and one L chain.

In the present invention, the antibody is preferably a Fab fragment.

The method of the present invention includes a step of obtaining atranslation product containing a target component-nucleic acid complexcontaining the target component and the nucleic acid encoding the targetcomponent, by translating a nucleic acid encoding any one component ofinterest (target component) among components constituting a proteinmultimer into the target component by an in vitro translation system(step i) or step i′)). For example, when an antibody-nucleic acidcomplex containing the antibody, and the nucleic acid encoding any onechain (target chain) (for example, L chain) selected from the groupconsisting of H chain and L chain constituting the antibody is to beproduced by the method of the present invention, a nucleic acid encodingthe target chain is translated into the target chain to give atranslation product containing the target chain-nucleic acid complexcontaining the target chain, and the nucleic acid encoding the targetchain.

In the present specification, the “in vitro translation system” refersto a protein synthesis system using a reaction mixture comprisingfactors necessary for protein synthesis such as cell extract and thelike, without the need for living cells, and is also referred to as acell-free protein synthesis system. That is, the in vitro translationsystem is characterized in that the living cells are not required intranslating an mRNA into a protein. The in vitro translation system inthe present invention includes a system for performing translation and asystem for performing transcription and translation. Hence, the in vitrotranslation system of the present invention encompasses any of thefollowing embodiments:

(1) translating an mRNA into a protein; and

(2) transcribing a DNA into an mRNA, and further translating the mRNAinto a protein.

The in vitro translation system includes an in vitro translation systemusing a cell extract of Escherichia coli, wheat germ, rabbitreticulocyte, cultured cell and the like, and a reconstituted in vitrotranslation system constituted from independently purified factors. Thein vitro translation system to be used in the present invention ispreferably a reconstituted in vitro translation system constituted fromindependently purified factors. Preferable examples of the reconstitutedin vitro translation system include a PURE system (JP-B-4061043,JP-A-2009-112286, Y. Shimizu et al., (2001) Nat. Biotechnol., vol. 19,p. 751-755, JP-A-2008-271903, E. Osada et al., (2009) J. Biochem., vol.145, p. 693-700). This reconstituted in vitro translation system canprevent contamination of nuclease and protease more easily than the invitro translation system using a cell extract, and therefore, canimprove the efficiency of the translation from a nucleic acid encoding atarget component into the target component. In addition, a targetcomponent-nucleic acid complex containing the target component and thenucleic acid encoding the target component can be stably formed andmaintained. Finally, therefore, the desired protein multimer-nucleicacid complex can be obtained efficiently.

In the present specification, the “factors” refers to a building blockof an in vitro translation system that can be purified independently.The factors include single proteins and substrates. Furthermore, variouscomplexes and mixtures that can be isolated from a crude fraction arealso included. For example, factors to be purified as a complex includemultimer of proteins, ribosomes and the like. Mixtures include tRNAmixture and the like. The “independently purified factors” refer tofactors that have been each independently purified from other factors.It is possible to reconstitute and construct an in vitro translationsystem by mixing as required the independently purified factorsnecessary for protein synthesis. Factors that present in a mixedfraction containing a plurality of kinds of factors without beingisolated from the cell extract are not said to be independently purifiedfactors. Even in the case of a complex consisting of a plurality ofcomponents, the complex is “independently purified factors” in thepresent specification, provided that it has been purified as singlefactors. For example, a purified ribosome is a complex consisting ofseveral elements, and it is an “independently purified factor” becauseit can be purified as a single factor.

Independently purified factors can be obtained by purifying fromextracts of a wide variety of cells. Cells for purifying the factorsinclude, for example, prokaryotic cells and eukaryotic cells.Prokaryotic cells include Escherichia coli cells, extreme-thermophilicbacterial cells, and Bacillus subtilis cells. Eukaryotic cells includeyeast cells, plant cells, insect cells, and mammalian cells. Inparticular, when the independently purified factors consist of a proteinonly, each factor can be obtained by one of the methods shown below.

(1) Isolating a gene that encodes each factor (protein) and introducinginto an expression vector, after which an appropriate host cell istransformed therewith to express the factor, and the expressed factor isrecovered.

(2) Isolating a gene that encodes each factor, synthesizing the factorusing an in vitro translation system, and recovering it. In (1), first,an expression plasmid is prepared by inserting the gene for each factorinto an expression vector comprising an expression regulatory region sothat the desired factor will be expressed under the control of theregion. An expression regulatory region that constitutes the vectorrefers to, for example, an enhancer, a promoter, a terminator and thelike. The expression vector can comprise a drug resistance marker andthe like. Next, host cells are transformed with this expression plasmidto allow each factor to be expressed.

The in vitro translation system preferably used for the method of thepresent invention consists of purified factors. In the presentinvention, the in vitro translation system can contain, for example, thefollowing factors in an independently purified state:

an initiation factor (IF),

an elongation factor (EF),

a release factor (RF),

an aminoacyl-tRNA synthetase (AARS),

a ribosome,

an amino acid,

a nucleoside triphosphate, and

a tRNA.

These factors are not limited to those derived from prokaryotic cellssuch as Escherichia coli, and those derived from eukaryotic cells canalso be used.

By adding the factors that constitute the above-described in vitrotranslation system to a buffer solution that maintains a pH suitable forthe transcription and translation, a composition having an in vitrotranslation activity can be obtained. Suitable pH is, for example, pH 6to pH 9, preferably pH 7 to 8. Buffer solutions which can be used in thepresent invention include potassium phosphate buffer solution (pH 7.3),Hepes-KOH (pH 7.6) and the like.

A salt can also be added to the composition having an in vitrotranslation activity for the purpose of protecting factors andmaintaining the activity thereof. Specifically, potassium glutamate,potassium acetate, ammonium chloride, magnesium acetate, magnesiumchloride, calcium chloride and the like can be mentioned.

Other low-molecular weight compounds can be added to the compositionhaving an in vitro translation activity as an enzyme substrate and/orfor the purpose of improving and maintaining the activity of eachfactor. Specifically, polyamines such as putrescine and spermidine,reducing agents such as dithiothreitol (DTT) and the like can be addedto the composition.

In the case of an in vitro translation system using factors derived fromprokaryotic cells such as of Escherichia coli, it is preferable that acomposition having an in vitro translation activity further comprisesribosome recycling factor (RRF), methionyl-tRNA transformylase (MTF) and10-formyl 5,6,7,8-tetrahydrofolate (FD).

When a DNA is transcribed into an mRNA and the mRNA is translated into apolypeptide, a composition having an in vitro translation activity cancontain an RNA polymerase for transcription to the mRNA. Specifically,the RNA polymerases shown below can be utilized in the presentinvention. These RNA polymerases are commercially available.

T7 RNA polymerase

T3 RNA polymerase

SP6 RNA polymerase

The composition having an in vitro translation activity can comprise, inaddition to factors for transcription and translation, an additionalfactor. Additional factors include, for example, the factors shownbelow.

Enzymes for recycling energy in the reaction system:

creatine kinase,

myokinase, and

nucleoside diphosphate kinase and the like;

substrates for enzymes for recycling energy in the reaction system:

creatine phosphate and the like;

enzymes for degradating inorganic pyrophosphoric acid resulting fromtranscription/translation:

inorganic pyrophosphatase and the like.

In the composition having an in vitro translation activity, it ispreferable that at least one of the independently purified factors beextracted from a prokaryote. In one embodiment, at least one, preferablyall, selected from the group consisting of an initiation factor, anelongation factor, a release factor, an aminoacyl-tRNA synthetase, aribosome and a tRNA, contained in the composition having an in vitrotranslation activity have been extracted from a prokaryote (for example,a Gram-negative bacterium, preferably Escherichia coli). In oneembodiment, the composition having an in vitro translation activityconsists of independently purified factors, and all of the factors areextracted from a prokaryote (for example, a Gram-negative bacterium,preferably Escherichia coli).

The constitution of the composition having an in vitro translationactivity can be adjusted as appropriate according to the kind of theprotein to be synthesized, as well as to the above-described basicconstitution. For example, in the case of a protein unlikely to form ahigher-order structure, an in vitro translation system supplemented witha class of proteins called molecular chaperones can be used.Specifically, an in vitro translation system supplemented with Hsp100,Hsp90, Hsp70, Hsp60, Hsp40, Hsp10, small Hsp, a homologue thereof,Escherichia coli trigger factor and the like can be mentioned. Molecularchaperones are proteins known to assist protein folding to form ahigher-order structure in cells to prevent the protein from aggregating(Bukau and Horwich, Cell (1998) vol. 92, p. 351-366, Young et al., Nat.Rev. Mol. Cell Biol (2004) vol. 5, p. 781).

When a disulfide bond is formed between components of a protein multimeror in the molecules of a component, like antibody, the redox potentialof the reaction mixture is important. For this reason, the reducingagent DTT may be removed from the reaction mixture, or a compositionsupplemented with a reagent that controls the redox potential such asoxidized glutathione may be used. Furthermore, it is possible to use acomposition supplemented with an enzyme that promotes the formation ofdisulfide bond, or isomerizes the disulfide bond correctly.Specifically, such enzymes include protein disulfide isomerase (PDI),which is present in the endoplasmic reticulum of eukaryotic cells, DsbAfrom Escherichia coli, DsbC and the like.

The purification methods of each factor contained in the reconstitutedcomposition having an in vitro translation activity and more detailedconstitution of the composition having an in vitro translation activityare described in JP-A-2008-271903, JP-A-2003-10249 and the like, and areknown to those of ordinary skill in the art. In addition, compositionshaving the in vitro translation activity are commercially available fromplural companies and can be obtained easily.

A translation product containing a target component-nucleic acid complexcontaining the target component and the nucleic acid encoding the targetcomponent can be obtained by adding the nucleic acid encoding the targetcomponent to the above-mentioned composition having an in vitrotranslation activity, and incubating the mixture at a temperature atwhich the in vitro translation proceeds, thus translating the nucleicacid into the target component. While the incubation time is notparticularly limited as long as it is sufficient for completion of thetranslation of the nucleic acid into the target component, it isgenerally about 5-240 min.

What is important here is that the in vitro translation in steps i) andi′) is performed in the absence of a nucleic acid encoding a non-targetcomponent constituting the protein multimer together with the targetcomponent. Thus, a protein multimer-nucleic acid complex can be producedhighly efficiently by first performing the translation into a targetcomponent, and thereafter, in the presence of the translation product,performing the translation into a non-target component, or by performingthe translation into the target component and that into the non-targetcomponent in separate reaction systems, and thereafter mixing alltranslation products.

In the present specification, the “nucleic acid” refers mainly to apolymer of a deoxyribonucleotide or ribonucleotide. Hence, the nucleicacid is a deoxyribonucleic acid (DNA) or a ribonucleic acid (RNA).Furthermore, the nucleic acids in the present invention can comprisenucleotide derivatives having a non-natural base. The nucleic acids canalso comprise peptide nucleic acids (PNAs). As far as a translation intoan encoded target component can be performed by an in vitro translationsystem, the building block of the nucleic acid may be any one of thesenucleic acids or a mixture thereof. Therefore, DNA-RNA hybridnucleotides are included in the nucleic acids in the present invention.Alternatively, a chimeric nucleic acid generated by connecting differentnucleic acids, like DNA and RNA, into a single strand is also includedin the nucleic acids in the present invention. The structure of anynucleic acid in the present invention is not limited, as far as atranslation into an encoded target component can be performed by an invitro translation system. Specifically, the nucleic acid can assume astructure such as a single strand, double strand, or triple strand. Thenucleic acid to be used in the method of the present invention ispreferably an mRNA or cDNA encoding the target component.

When a prokaryote (such as Escherichia coli and the like)-derivedribosome is utilized in an in vitro translation system, a nucleic acidencoding the target component preferably contains the Shine-Dalgarno(SD) sequence, which is a ribosome-binding sequence, at the upstream ofthe initiation codon. When the SD sequence is contained, the efficiencyof the translation increases.

To form a stable target component-nucleic acid complex containing thetarget component, and the nucleic acid encoding the target component, itis preferable to modify the nucleic acid encoding the target component.As a technique to form a stable protein-nucleic acid complex, ribosomedisplay (Proc. Natl. Acad. Sci. USA, 91, 9022-9026, 1994; Proc. Natl.Acad. Sci. USA, 94, 4937-4942, 1997; JP-A-2008-271903), mRNA display(FEBS Lett., 414.405-408, 1997), CIS display (Proc. Natl. Acad. Sci.USA, 101, 2806-2810, 2004), Covalent antibody display (Nucleic AcidsRes., 33(1), e10, 2005) and the like can be mentioned. The constitutionof the target component-nucleic acid complex formed changes according tothe modification of the nucleic acid encoding the target component.

In ribosome display (Proc. Natl. Acad. Sci. USA, 91, 9022-9026, 1994;Proc. Natl. Acad. Sci. USA, 94, 4937-4942, 1997; JP-A-2008-271903), thenucleic acid (preferably mRNA) encoding the target component preferablyhas at least one (preferably two, more preferably all) of the followingcharacteristics to improve the selection efficiency.

(1) Containing a sequence encoding a spacer at the downstream of thetarget gene.

(2) Containing the arrest sequence such as partial sequence of SecM andthe like at the downstream of the spacer.

(3) Removing a stop codon.

In ribosome display, a sequence that encodes a spacer is preferablycontained at the downstream of the gene of the target component. Thespacer prevents steric hindrance between the newly generated polypeptideand the ribosome by providing a sufficient space for the translatedpolypeptide to be accurately folded outside of the ribosome. Here,without a spacer of sufficient length, the desired polypeptide (targetcomponent) is unable to go out completely from the ribosome, so thatselection by ribosome display cannot be performed efficiently. Thespacer consists of at least 20 amino acids, preferably 30 amino acids ormore, more preferably 40 amino acids or more in length. Specifically, apartial sequence of the phage gene III, a partial sequence ofEscherichia coli tolA and the like can be used.

Furthermore, a nucleic acid (preferably, mRNA) having a sequence thatencodes Escherichia coli SecM translation elongation arrest sequence(arrest sequence; amino acid residues 148 to 170) placed downstream ofthe spacer sequence is preferably used. This translation elongationarrest sequence has been shown to firmly interact with the peptidetunnel in ribosome (Nakatogawa et al., Cell (2002) vol. 108, p.629-636), and has been proven to efficiently stop the elongation of thetranslation when using a reconstituted in vitro translation system (Mutoet al., Mol. Cell (2006) vol. 22, p. 545-552). A stable targetcomponent-ribosome-mRNA ternary complex can be formed by tightinteraction of ribosome and translation elongation arrest sequence ofSecM.

A nucleic acid (preferably, mRNA) with such a structure can be obtainedby, for example, inserting the gene of the target component into anexpression vector harboring a 5′ UTR sequence comprising a promotersequence and the SD sequence, or a 3′ terminal spacer sequence, andtranscribing the same using RNA polymerase. Generally, RNA polymeraserecognizes a region comprising a particular sequence called a promoter,and synthesizes an mRNA on the basis of the nucleic acid sequence of theDNA placed downstream thereof. It is also possible to construct atranscription template having the desired structure by utilizing PCRwithout using an expression vector (Split-Primer PCR method, Sawasaki etal., PNAS (2002) vol. 99, p. 14652-14657). In this method, a templateDNA is constructed by adding a 5′ UTR sequence and a spacer sequence tothe desired DNA by PCR. In preparing an mRNAs library from a DNAlibrary, it is unnecessary to clone a DNA into the above-describedvector. For this reason, time and labor can be saved.

How to construct a template DNA by PCR is specifically exemplifiedbelow.

(1) The DNA region that encodes the target component is amplified froman appropriate library and the like by a PCR using a primer comprising a5′ UTR sequence comprising a promoter and the SD sequences and a primercomprising a portion of a spacer sequence and removing a stop codon.(2) The amplified DNA is again amplified with the primer for the 5′ UTRportion and a primer comprising a spacer portion and SecM sequence.

By further amplifying the DNA thus constructed as required, andperforming transcription using RNA polymerase with the amplified DNA asthe template, an mRNA that serves as the template for the translationcan be obtained.

The mRNA transcribed by RNA polymerase is recovered as required, andadded to a composition having an in vitro translation activity. Thetranscribed mRNA can be recovered by ethanol precipitation after phenoltreatment. Commercially available RNA extraction kits such as RNeasy(manufactured by Qiagen) can be utilized for recovering the mRNA.

Also, the above-described DNA itself comprising the nucleic acidsequences necessary for the transcription and translation incorporatedin the gene of the target component can also be used as the template. Inthis case, the DNA is transcribed into an mRNA using the compositionhaving an in vitro translation activity comprising RNA polymerase, andthe mRNA is translated into a polypeptide to form the ternary complex.

In mRNA display (FEBS Lett., 414.405-408, 1997), puromycin is added tothe 3′-terminal of the nucleic acid (preferably mRNA) encoding thetarget component. Puromycin can be added to a nucleic acid (preferablymRNA) by a method utilizing UV crosslinkage (Nucleic Acids Res., 28,e83, 2000) or a method utilizing hybridization of primer (FEBS Lett.,508, 309-312, 2001). By subjecting a nucleic acid (preferably mRNA)encoding a target component, wherein puromycin is added to the 3′terminus, to an in vitro translation, a complex, wherein the translatedtarget component and the nucleic acid (preferably, mRNA) are covalentlybound via puromycin, can be formed.

In the nucleic acid (preferably mRNA) encoding the target component tobe used for CIS display (Proc. Natl. Acad. Sci. USA, 101, 2806-2810,2004), RepA gene is fused in-frame to the 3′ terminal of the codingregion of the target component, and the CIS element is further fused tothe downstream thereof. By in vitro translation using this nucleic acidas a template, a fusion protein of the target component and RepA isproduced, and RepA contained in the fusion protein binds to the CISelement, thus forming a complex wherein the target component and thenucleic acid encoding the target component are linked via RepA.

In the nucleic acid encoding the target component (preferably DNA) to beused for the covalent antibody method (Nucleic Acids Res, 33(1), e10,2005), endonuclease P2A gene is fused in-frame to the 3′ end of thecoding region of the target component via a spacer. By translation usingthe nucleic acid as a template, a fusion protein of the target componentand P2A is produced, and P2A contained in the fusion protein iscovalently bound to the DNA encoding the fusion protein, thus forming acomplex wherein the target component and the nucleic acid encoding thetarget component are linked via P2A.

In the first embodiment, a protein multimer-nucleic acid complexcomprising the protein multimer and the nucleic acid encoding the targetcomponent is obtained by adding a nucleic acid encoding a non-targetcomponent constituting the protein multimer together with the targetcomponent to the translation product obtained in step i) to performtranslation of the nucleic acid encoding the non-target component intothe non-target component to give the non-target component, andassociating the non-target component with the target component containedin the target component-nucleic acid complex to form a protein multimer(step ii). For example, when an antibody-nucleic acid complex containingan antibody, and a nucleic acid encoding any one chain (target chain)(for example, L chain) selected from the component group consisting of Hchain and L chain constituting the antibody is to be produced by themethod of the present invention, a nucleic acid encoding a non-targetchain (for example, H chain) constituting the antibody together with thetarget chain is added to translate the nucleic acid encoding thenon-target chain into the non-target chain to give the non-target chain,and the non-target chain is associated with the target chain containedin the target chain-nucleic acid complex to form the antibody, wherebythe antibody-nucleic acid complex containing the antibody, and thenucleic acid encoding the target chain is obtained.

The translation product obtained in step i) is expected to retain the invitro translation activity even after completion of the translation instep i). Therefore, when a nucleic acid encoding a non-target componentis added to the translation product obtained in step i), the nucleicacid is translated into the non-target component due to the remaining invitro translation activity.

In the second embodiment, separately from step i′), a nucleic acidencoding a non-target component constituting the protein multimertogether with the target component is translated into the non-targetcomponent by an in vitro translation system to give a translationproduct containing the non-target component (step ii′), and thetranslation product of i′) is further mixed with the translation productof ii′), and the non-target component is associated with the targetcomponent contained in the target component-nucleic acid complex to forma protein multimer, whereby the protein multimer-nucleic acid complexcomprising the protein multimer and the nucleic acid encoding the targetcomponent is obtained (step iii′). For example, when an antibody-nucleicacid complex containing an antibody, and a nucleic acid encoding any onechain (target chain) (for example, L chain) selected from the componentgroup consisting of H chain and L chain constituting the antibody is tobe produced by the method of the present invention, separately from thetranslation of the target chain, a nucleic acid encoding a non-targetchain (for example, H chain) constituting the antibody together with thetarget chain is translated into the non-target chain by an in vitrotranslation system to give a translation product containing thenon-target chain (for example, H chain), the translation productcontaining the target chain (for example, L chain)-nucleic acid complexand the translation product containing the non-target chain (forexample, H chain) are mixed, and the non-target chain is associated withthe target chain contained in the target chain-nucleic acid complex toform the antibody, whereby the antibody-nucleic acid complex containingthe antibody, and the nucleic acid encoding the target chain isobtained.

The nucleic acid encoding the non-target component may be mRNA or cDNA.When the in vitro translation system used in step i) of the firstembodiment does not contain an RNA polymerase to transcribe DNA to mRNA,the nucleic acid encoding the non-target component is preferably mRNA.This is because translation into a non-target component is possible evenwithout newly adding an RNA polymerase. On the other hand, when the invitro translation system used in step i) contains an RNA polymerase totranscribe DNA to mRNA, the nucleic acid encoding the non-targetcomponent may be any of mRNA and cDNA.

To improve the translation efficiency of a non-target component, when aribosome derived from prokaryote such as Escherichia coli and the likeis utilized in an in vitro translation system, a nucleic acid(preferably mRNA) encoding the non-target component preferably containsthe SD sequence at the upstream of the initiation codon.

Since the nucleic acid encoding the non-target component does not needto be corresponded to the non-target component, modification of thenucleic acid therefor (addition of spacer to the downstream of thetarget gene, partial sequence of SecM to the downstream of spacer etc.)is not necessary.

Among the factors contained in the composition having an in vitrotranslation activity used in step i) of the first embodiment, thefactor(s) consumed in the translation in step i) may be added to thetranslation product obtained in step i) together with the nucleic acidencoding the non-target component. As such factor, ribosome can bementioned. Particularly, when a target component-ribosome-nucleic acid(preferably mRNA) ternary complex is formed in step i), the ribosome maybe introduced into the ternary complex without being recycled anddepleted from the in vitro translation system, and therefore, it ispreferable to add, in step ii), ribosome to the translation productobtained in step i). Ribosome is added to the translation productobtained in step i) such that the concentration excluding ribosomecontained in the above-mentioned ternary complex is, for example, 0.01μM-50 μM preferably 0.05 μM-10 μM.

In step ii), the non-target component is produced by adding the nucleicacid encoding the non-target component to the translation productobtained in step i) and incubating the mixture at a temperature at whichthe in vitro translation proceeds, thus translating the nucleic acidinto the non-target component. While the incubation time is notparticularly limited as long as it is sufficient for completion of thetranslation of the nucleic acid into the non-target component, it isgenerally about 5-240 min. The produced non-target component isassociated with the target component contained in the targetcomponent-nucleic acid complex in the reaction mixture to form a proteinmultimer, whereby a protein multimer-nucleic acid complex containing theprotein multimer, and the nucleic acid encoding the target component canbe obtained.

The embodiment of the composition having an in vitro translationactivity, which is used in step ii′) of the second embodiment is asdescribed above as the embodiment of the composition having an in vitrotranslation activity, which is used in step i′).

In step ii′), a translation product containing the non-target componentcan be produced by adding the nucleic acid encoding the non-targetcomponent to the above-mentioned composition having an in vitrotranslation activity and incubating the mixture at a temperature atwhich the in vitro translation proceeds, thus translating the nucleicacid into the non-target component. The incubation time is notparticularly limited as long as it is sufficient for completion of thetranslation of the nucleic acid into the non-target component, it isgenerally about 5-240 min.

What is important here is that the in vitro translation in steps ii′) isperformed in the absence of the nucleic acid encoding the targetcomponent. Thus, a protein multimer-nucleic acid complex can be producedhighly efficiently by performing the translation into the targetcomponent and that into the non-target component separately in differentreaction systems, and thereafter mixing all translation products.

The protein multimer-nucleic acid complex comprising the proteinmultimer and the nucleic acid encoding the target component is obtainedby further mixing the translation product of i′) with the translationproduct of ii′), associating the non-target component with the targetcomponent contained in the target component-nucleic acid complex to formthe protein multimer (step iii′).

When a translation product containing a target component-ribosome-mRNAternary complex is obtained by ribosome display in step i) or i′), aprotein multimer-ribosome-mRNA ternary complex containing a proteinmultimer, mRNA encoding a target component and ribosome is obtained instep ii) or iii′).

When a translation product containing a complex wherein the targetcomponent and the nucleic acid (preferably, mRNA) encoding the targetcomponent are covalently linked via puromycin is obtained by mRNAdisplay in step i) or i′), a complex wherein the protein multimer andthe nucleic acid (preferably, mRNA) encoding the target component arecovalent linked via puromycin is obtained in step ii) or iii′).

When a translation product containing a complex wherein the targetcomponent and the nucleic acid encoding the target component are linkedvia RepA is obtained by CIS display in step i) or i′), a complex whereinthe protein multimer and the nucleic acid encoding the target componentare linked via RepA is obtained in step ii) or iii′).

When a translation product containing a complex wherein the targetcomponent and the nucleic acid encoding the target component are linkedvia P2A is obtained by a covalent antibody method in step i) or i′), acomplex wherein the protein multimer and the nucleic acid encoding thetarget component are linked via P2A is obtained in step ii) or iii′).

A protein multimer-nucleic acid complex produced by the method of thepresent invention contains one molecule of the protein multimer and onemolecule of the nucleic acid encoding the target component, per moleculeof the complex. That is, in the complex, since the protein multimercorresponds one-to-one to the nucleic acid encoding the component in theprotein multimer, the nucleic acid encoding the component contained inthe object protein multimer can be obtained by selecting a ternarycomplex containing a protein multimer having a desired activity such asspecific binding to a particular target substance and the like andamplifying the nucleic acid contained in the ternary complex.

Although not restricted by theory, when a target component-nucleicacid-ribosome ternary complex containing the target component, thenucleic acid encoding the target component and ribosome is formed bytranslating the nucleic acid encoding the target component into thetarget component by ribosome display, ribosome turnover is suppressed,since ribosome is stably incorporated into the ternary complex. Incontrast, the ribosome turnover actively occurs in the translation ofthe non-target component, since incorporation of ribosome into thecomplex does not occur. Therefore, when translation of the targetcomponent and that of the non-target component, showing oppositeribosome turnover conditions, are simultaneously performed in the sametube, regulation of ribosome may become inconsistent and the finalproduct amount of the protein multimer-ribosome-nucleic acid ternarycomplex may decrease. In contrast, in the method of the presentinvention, the target component is first translated under an environmentsuppressing ribosome turnover to form a target component-nucleicacid-ribosome ternary complex, after which a non-target component istranslated under a high ribosome turnover environment in the presence ofa target component-nucleic acid-ribosome ternary complex (the firstembodiment), or in a tube separated from the tube for the translation ofthe target component (the second embodiment). As a result, regulation ofribosome becomes consistent, and smooth and accurate association of thetarget component and the non-target component occurs. Consequently,there is a high possibility that a protein multimer-ribosome-nucleicacid ternary complex is formed with high efficiency.

In one aspect, a library of the nucleic acid encoding the targetcomponent can be used as the nucleic acid encoding the target component.In this case, in the first embodiment, the library of a proteinmultimer-nucleic acid complex comprising a protein multimer and anucleic acid encoding a target component can be obtained by

i) translating a library of a nucleic acid encoding the target componentinto the target component by an in vitro translation system to give atranslation product containing a library of a target component-nucleicacid complex containing the target component and the nucleic acidencoding the target component, andii) translating a nucleic acid encoding a non-target componentconstituting the protein multimer together with the target componentinto the non-target component by adding the nucleic acid encoding thenon-target component to the translation product obtained in i) to givethe non-target component, associating the non-target component with thetarget component in the target component-nucleic acid complex to formthe protein multimer.

In the second embodiment, a library of a protein multimer-nucleic acidcomplex comprising a protein multimer and a nucleic acid encoding thetarget component can be obtained by

i′) translating a library of a nucleic acid encoding the targetcomponent into the target component by an in vitro translation system togive a translation product containing a library of a targetcomponent-nucleic acid complex containing the target component and thenucleic acid encoding the target component,ii′) translating a nucleic acid encoding a non-target componentconstituting the protein multimer together with the target component byan in vitro translation system to give a translation product containingthe non-target component, andiii′) mixing the translation product of i′) with the translation productof ii′), associating the non-target component with the target componentcontained in the target component-nucleic acid complex to form a proteinmultimer.

The present invention also provides a method of producing such libraryof a protein multimer-nucleic acid complex comprising a protein multimerand a nucleic acid encoding any one target component of the proteinmultimer.

In the present invention, the “library” refers to a population withdiversity, which consists of a plurality of cloned nucleic acids. Anucleic acid that encodes a target component contained in a proteinmultimer having a desired property can be obtained from a library usingan in vitro selection system such as ribosome display. The library ofnucleic acids usable in the present invention includes a cDNA library,an mRNA library, and a genomic DNA library. In prokaryotic cells andyeast cells, usually no intron is present in most genes. Therefore, inthe case of prokaryotic cells and yeast cells, a genomic DNA library canbe utilized to directly screen for a nucleic acid that encodes a proteinhaving the desired property from proteins derived from the cells. Inhigher eukaryotes such as mammals, conversely, an intron is present inmost genes, so that an mRNA library or a cDNA library is usuallyutilized.

The sequences of nucleic acids that constitute the library can comprisenot only sequences of natural origin, but also artificially introducedsequences. For example, libraries incorporating mutations are includedin the library in the present invention. Alternatively, a librarycomprising sequences prepared by joining an artificial sequence to asequence of natural origin is also included in the library in thepresent invention. Furthermore, a library comprising a completelyartificially designed sequence is also included in the library in thepresent invention.

Moreover, by contacting the complex obtained by step ii) or step iii′)with a target substance, and evaluating the affinity of the proteinmultimer contained in the complex for the target substance, a proteinmultimer having affinity for the desired target substance can beselected and the nucleic acid sequence of the target component containedin the protein multimer can be identified. In the present invention, the“target substance” refers to a substance to which the desired proteinmultimer can bind. In the present invention, any substance to which aprotein multimer may bind can be utilized as the target substance. Thetarget substances of the present invention include, for example, nucleicacids, polypeptides, organic compounds, inorganic compounds,low-molecular weight compounds, sugar chains, fats, and lipids. Morespecifically, a substance that functions as an antigen or hapten can beutilized as the target substance. In this case, the desired antibody canbe screened from an antibody library, and the sequence of the nucleicacid encoding one of the L chain and the H chain contained in theantibody can be determined.

Conditions for contacting a protein multimer-nucleic acid complex with atarget substance to enable their binding are publicly known (WO95/11922,WO93/03172, WO91/05058), and can be established without excess burdensby those skilled in the art. In order to recover the complex bound tothe target substance, it is necessary to screen for the complex bound tothe target substance from among complexes not bound to the targetsubstance. This is performed according to a known method called panning(Coomber, Method Mol. Biol. (2002) vol. 178, p. 133-145). The basicprocedures for panning are as described below.

(1) The complex is contacted with the target substance immobilized on asolid phase carrier. Alternatively, the complex is contacted with thetarget substance labeled with a binding partner to be captured by asolid phase carrier, after which the target substance bound to thecomplex is immobilized onto the solid phase carrier.(2) The complexes not bound to the target substance are removed. Forexample, the same can be removed by washing.(3) The complex that has not been removed is recovered.(4) The processes (1) to (3) are repeated a plurality of times asrequired.

When repeating the series of steps, it is also possible to amplify themRNA that constitutes the recovered complex before the step (1). ThemRNA can be amplified by, for example, RT-PCR. DNA is synthesized byRT-PCR with the mRNA as the template. The DNA may be transcribed againto an mRNA, and can be utilized for forming the complex. For mRNAtranscription, DNA can be inserted into a vector. Alternatively, thestructure necessary for the transcription may be joined to DNA totranscribe to mRNA.

In the present specification, “screening” refers to selecting an entitywith the desired property from among substances synthesized by achemical synthesis, an enzymatic reaction or a combination thereof,substances prepared from extracts of various cells, or naturallyoccurring substances. “Cloning” refers to isolating a particular gene.

After the complex that presents the desired protein multimer isselected, the sequence of the nucleic acid that encodes the targetcomponent contained in the complex can be identified. At the stage whenthe complex is selected, the nucleic acid that encodes the targetcomponent is an mRNA. By synthesizing a cDNA using a reversetranscriptase with this mRNA as the template, and reading the nucleicacid sequence using a sequencer, the nucleic acid sequence thereof canbe determined. These techniques are publicly known.

The contents disclosed in any publication cited herein, includingpatents and patent applications, are hereby incorporated in theirentireties by reference, to the extent that they have been disclosedherein.

The present invention is explained in more detail in the following byreferring to Examples, which are not to be construed as limitative.

EXAMPLES Example 1

<Method>

Construction of Anti-TNF-α Antibody Gene for Ribosome Display

The genes of the H and L chains of an anti-TNF-α antibody werechemically synthesized (GenScript) by reference to JP-B-3861118, and atthat time, FLAG tag sequence+stop codon was added to the 3′ terminus ofthe H chain, and c-Myc tag sequence was added to the 3′ terminus of theL chain.

H Chain:

(SEQ ID NO: 1) ATGGAGGTGCAATTGGTGGAGTCTGGGGGAGGCTTGGTACAGCCCGGCAGGTCCCTGAGACTCTCCTGTGCGGCCTCTGGATTCACCTTTGATGATTATGCCATGCACTGGGTCCGGCAAGCTCCAGGGAAGGGCCTGGAATGGGTCTCAGCTATCACTTGGAATAGTGGTCACATAGACTATGCGGACTCTGTGGAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAACTCCCTGTATCTGCAAATGAACAGTCTGAGAGCTGAGGATACGGCCGTATATTACTGTGCGAAAGTCTCGTACCTTAGCACCGCGTCCTCCCTTGACTATTGGGGCCAAGGGACCCTGGTCACCGTCTCGAGTGCTAGCTTCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAGAGTTGAGCCCAAATCTGAATTCGACTATAAAGATGACGATGACA AATAATGAL Chain:

(SEQ ID NO: 2) ATGGATATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGGGACAGAGTCACCATCACTTGTCGGGCAAGTCAGGGCATCAGAAATTACTTAGCCTGGTATCAGCAAAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATGCTGCATCCACTTTGCAATCAGGGGTCCCATCTCGGTTCAGTGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGCCTACAGCCTGAAGATGTTGCAACTTATTACTGTCAAAGGTATAACCGTGCACCGTATACTTTTGGCCAGGGGACCAAGGTGGAAATCAAACGAACTGTGGCGGCGCCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCTCGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGATTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGGAGCAGAAGCTGATCTCTGAGGAGGATCTGCAT

In addition, 5′ UTR sequence containing T7 promoter and the SD sequencenecessary for the expression with in vitro translation system fromEscherichia coli were also synthesized chemically (Sigma).

H Chain 5′ UTR:

(SEQ ID NO: 3) gaaattaatacgactcactatagggagaccacaacggtttccctctagaaataattttgtttaactttaagaaggagatataccaATGGAGGTGCAATTG GTGGAGTCTGGGGGAGL Chain 5′ UTR:

(SEQ ID NO: 4) gaaattaatacgactcactatagggagaccacaacggtttccctctagaaataattttgtttaactttaagaaggagatataccaATGGATATCCAGATGACCCAGTCTCCATCCTCCCTG

A partial sequence (the 220-326th amino acid residues) of geneIII (g3p)of M13 phage was amplified by PCR using M13KO7-phage genome as atemplate and the following primer set, with KOD Plus DNA Polymerase(TOYOBO) (denaturation: 94° C., 10 sec, annealing: 58° C., 30 sec,extension: 68° C., 60 sec, cycles: 25), and the product was purifiedusing a QIAquick PCR purification kit (QIAGEN).

Primer Myc-g3p:

(SEQ ID NO: 5) GAGCAGAAGCTGATCTCTGAGGAGGATCTGCATGAATATCAAGGCCAATCGTCTGAC Primer g3p-SecMstop:

(SEQ ID NO: 6) CTCGAGTTATTCATTAGGTGAGGCGTTGAGGGCCAGCACGGATGCCTTGCGCCTGGCTTATCCAGACGGGCGTGCTGAATTTTGCGCCGGAAACGTCACC AATGAAAC 

A PCR reaction mixture (total 500 μL) containing respectivechemically-synthesized gene fragments (H chain 5′UTR and H chain, each 1pmol), 5′ primer:

(SEQ ID NO: 7, 10 pmol)GAAATTAATACGACTCACTATAGGGAGACCACAACGGTTTCCCTCTAG, FLAG stop R:

(SEQ ID NO: 8, 10 pmol) TCATTATTTGTCATCGTCATCTTTATAGTCG,and KOD Plus DNA Polymerase (TOYOBO) was prepared, and 25 cycles of PCRreaction (denaturation: 94° C., 10 sec, annealing: 58° C., 30 sec,extension: 68° C., 60 sec) were performed. A band in which two geneswere connected was confirmed by electrophoresis using 1% agarose, andthe band was excised and purified by a MinElute Gel Extraction Kit(QIAGEN) to finally give an H chain gene. In addition, in the samemanner, a PCR reaction solution (total 500 μL) containing respectivechemically-synthesized gene fragments (L chain 5′UTR and L chain) andg3p gene (each 1 pmol), 5′ primer:

(SEQ ID NO: 7, 10 pmol) GAAATTAATACGACTCACTATAGGGAGACCACAACGGTTTCCCTCTAG, primer SecMstop:

(SEQ ID NO: 9, 10 pmol) GGATTAGTTATTCATTAGGTGAGGCGTTGAGG,and KOD Plus DNA Polymerase (TOYOBO) was prepared, and 25 cycles of PCRreaction (denaturation: 94° C., 10 sec, annealing: 58° C., 30 sec,extension: 68° C., 60 sec) were performed. A band in which all geneswere connected was confirmed by electrophoresis using 1% agarose, andthe band was excised and purified by a MinElute Gel Extraction Kit(QIAGEN) to finally give an L chain gene.In Vitro Transcription

The purified H, L chain gene DNA (1 μg) was transcribed into mRNA by 20μL of in vitro transcription Kit (Ribomax™ Large Scale RNA ProductionSystem-T7, Promega), and purified by a column (RNeasy mini column,QIAGEN).

Formation of Ribosome Display Complex

The in vitro translation system (PURE system), which is a proteinsynthesis reaction reagent, was prepared according to a previous report(Shimizu et al. (2005) Methods, vol. 36, p 299-304).

(Condition 1)

To the prepared reaction mixture (10 μL) were added oxidized glutathione(GSSG: Sigma Ltd.) at a final concentration of 3 mM, Escherichia coliDsbC protein at a final concentration of 1 μM prepared in advance, Lchain-g3p mRNA (1 pmol), and H chain mRNA (1 pmol), and the mixture wasincubated at 30° C. for 60 min.

(Condition 2)

To the prepared reaction mixture (5 μL) were added oxidized glutathione(GSSG: Sigma Ltd.) at a final concentration of 3 mM, Escherichia coliDsbC protein at a final concentration of 1 μM prepared in advance, and Lchain-g3p mRNA (1 pmol), and the mixture was incubated at 30° C.Simultaneously, to the prepared reaction mixture (5 μL) were addedoxidized glutathione (GSSG: Sigma Ltd.) at a final concentration of 3mM, Escherichia coli DsbC protein at a final concentration of 1 μMprepared in advance, and H chain mRNA (1 pmol), and the mixture wasincubated at 30° C. Each was incubated for 30 min, respective reactionmixtures were mixed, and the mixture was further incubated at 30° C. for30 min.

(Condition 3)

To the prepared reaction mixture (10 μL) were added oxidized glutathione(GSSG: Sigma Ltd.) at a final concentration of 3 mM, Escherichia coliDsbC protein at a final concentration of 1 μM prepared in advance, and Lchain −g3p mRNA (1 pmol), and the mixture was incubated at 30° C. After30 min, H chain mRNA (1 pmol) and ribosome (5 pmol) were added, and themixture was further incubated at 30° C. for 30 min.

In respective conditions, 500 μL of ice-cooled Wash buffer (50 mMTris-OAc, pH 7.5, 150 mM NaCl, 25 mM Mg(OAc)₂, 0.5% Tween 20, 1 μg/mLSaccharomyces cerevisiae total RNA (Sigma) was added to the translationsolution to terminate the translation.

Biotinylation of Antigen Protein

Purchased TNF-α protein (TOYOBO) was biotinylated according to thestandard protocol for EZ-Link NHS-PEO₄-Biotin (PIERCE). Biotinylation ofeach biotinylated antigen protein was confirmed by the mobility shift ofthe band in SDS-PAGE, and the concentration was determined using a BCAProtein Assay Kit (PIERCE).

In Vitro Selection

Dynabeads MyOne streptavidin T1 magnetic beads (10 μL slurry,Invitrogen) blocked in advance with 5% SuperBlock (Thermo scientific) at4° C. overnight were washed twice with 500 μL of Wash buffer usingMagneSphere Magnetic Separation Stand (Promega), 1 nmol biotinylatedTNF-α protein was added, and immobilized on the magnetic beads at 4° C.After 30 min, the magnetic beads were washed three times with 500 μL ofWash buffer using MagneSphere Magnetic Separation Stand (Promega), thetranslation solution was added to the recovered magnetic beads, and themixture was stirred at 4° C. for 1 hr by rotation. The supernatant wasdiscarded using MagneSphere Magnetic Separation Stand (Promega), 1 mL ofWash buffer was added to the recovered magnetic beads, and the mixturewas stirred at 4° C. for 5 min by rotation. This operation was repeated30 times, 50 μL of Elution buffer (50 mM Tris-OAc, pH 7.5, 150 mM NaCl,50 mM EDTA) was added to the recovered magnetic beads, and the mixturewas left standing at 4° C. for 10 min to allow release of the complexfrom the magnetic beads. The supernatant was collected with MagneSphereMagnetic Separation Stand (Promega), and mRNA was collected and purifiedby RNeasy Micro (QIAGEN).

Real Time PCR

After in vitro selection, a reaction mixture containing the recoveredmRNA (1 μL), primer Realtime-F:

(SEQ ID NO: 10) GAGCAAAGCAGATTACGAGAAACAC,primer Myc-R:

(SEQ ID NO: 11) CAGATCCTCCTCAGAGATCAGC and RNA-direct SYBR Green Realtime PCR Master Mix (TOYOBO) was prepared,and the final mRNA amount was quantified according to the standardprotocol using LightCycler (Roche).<Results>

FIG. 1 shows comparison of the amounts of mRNA recovered by in vitroselection after translation under respective conditions 1 and 2, whereinthe amount under condition 2 was indicated as 100%. As a result, therewas about 5-fold difference in the mRNA recovery amount betweencondition 1 and condition 2. FIG. 2 shows comparison of the amounts ofmRNA obtained under respective conditions 1 and 3 in the same manner asin FIG. 1, wherein the amount under condition 3 was indicated as 100%.As a result, there was about 28-fold difference between condition 1 andcondition 3.

These results have clarified that a ribosome display complex containinga protein multimer can be formed at high efficiency, and the mRNArecovery rate after selection is drastically improved by

-   -   first forming a ribosome display complex for a library gene (L        chain of antibody here) in an in vitro translation system and        expressing a pair protein gene (H chain of antibody here) in the        same tube, or    -   forming a ribosome display complex for a library gene (L chain        of antibody here) and translating a pair protein gene (H chain        of antibody here) in separated tubes in an in vitro translation        system, and mixing all the translation products.

INDUSTRIAL APPLICABILITY

According to the present invention, a method of efficiently displaying aprotein multimer on a display complex so as to select a nucleic acidencoding a component contained in the protein multimer having a desiredfunction from the library is provided. By performing in vitro selectionsuch as ribosome display, mRNA display and the like using the presentinvention, the function such as binding affinity and the like of acomplicated protein multimer such as antibody and the like can beincreased or improved efficiently.

This application is based on U.S. provisional patent application No.61/638,774 (filing date: Apr. 26, 2012), the contents of which areencompassed in full herein.

The invention claimed is:
 1. An antibody-nucleic acid complex comprisingan antibody and a nucleic acid encoding any one target component of theantibody, wherein the antibody is a protein multimer containing at leasttwo chains including one H chain or an antigen binding portion thereofand one L chain or an antigen binding portion thereof, wherein thetarget component is selected from the group consisting of an H chain, anL chain, and antigen binding portions thereof, wherein, when the targetcomponent is the H chain or an antigen binding portion thereof, anon-target component is the L chain or an antigen binding portionthereof, wherein, when the target component is the L chain or an antigenbinding portion thereof, the non-target component is the H chain or anantigen binding portion thereof, and wherein the nucleic acid encodingthe target component does not encode the non-target component, andwherein the antibody-nucleic acid complex does not comprise a nucleicacid that encodes the non-target component.
 2. A library of theantibody-nucleic acid complex of claim
 1. 3. A method of producing theantibody-nucleic acid complex of claim 1, which comprises the followingsteps: i′) translating the nucleic acid encoding the target componentinto the target component by an in vitro translation system to give atranslation product containing a target component-nucleic acid complexcontaining the target component and the nucleic acid encoding the targetcomponent, wherein the nucleic acid does not encode the non-targetcomponent, ii′) providing a non-target component constituting theantibody together with the target component, and iii′) mixing thetranslation product of i′) with the non-target component of ii′),associating the non-target component with the target component containedin the target component-nucleic acid complex to form the antibody, thusaffording the antibody-nucleic acid complex that comprises the antibodyand the nucleic acid encoding the target component, wherein theantibody-nucleic acid complex does not comprises a nucleic acid encodingthe non-target component.
 4. The method according to claim 3, whereinthe in vitro translation system in i′) does not contain the nucleic acidencoding the non-target component.
 5. The method according to claim 3,wherein the antibody-nucleic acid complex and the targetcomponent-nucleic acid complex comprise a ribosome.
 6. The methodaccording to claim 3, wherein one molecule of the antibody, and onemolecule of the nucleic acid encoding the target component are containedper one molecule of the antibody-nucleic acid complex.
 7. The methodaccording to claim 1, wherein the in vitro translation system consistsof independently purified factors.
 8. The method according to claim 7,wherein at least one of the independently purified factors is a factorextracted from prokaryote.
 9. The method according to claim 1, whereinthe antibody consists of one H chain or an antigen binding portionthereof and one L chain or an antigen binding portion thereof.
 10. Themethod according to claim 1, wherein the antibody is a Fab fragment. 11.The method according to claim 1, wherein the nucleic acid encoding thetarget component is a library of the nucleic acid encoding the targetcomponent.
 12. The method according to claim 1, wherein the provision ofthe non-target component in ii′) is performed by translating a nucleicacid encoding the non-target component to give a translation productcontaining the non-target component.
 13. The method according to claim12, wherein the translation in ii′) is performed by an in vitrotranslation system.
 14. A method of producing the library of claim 2,which comprises the following steps: i′) translating a library of anucleic acid encoding the target component into the target component byan in vitro translation system to give a translation product containinga library of a target component-nucleic acid complex containing thetarget component and the nucleic acid encoding the target component,wherein the nucleic acid does not encode the non-target component, ii′)providing a non-target component constituting the antibody together withthe target component, and iii′) mixing the translation product of i′)with non-target component of ii′), associating the non-target componentwith the target component contained in the target component-nucleic acidcomplex to form the antibody, thus affording the library of theantibody-nucleic acid complex that comprises the antibody and thenucleic acid encoding the target component, wherein the antibody-nucleicacid complex does not comprises a nucleic acid encoding the non-targetcomponent.
 15. The method according to claim 14, wherein the provisionof the non-target component in ii′) is performed by translating anucleic acid encoding the non-target component to give a translationproduct containing the non-target component.
 16. The method according toclaim 15, wherein the translation in ii′) is performed by an in vitrotranslation system.
 17. The antibody-nucleic acid complex according toclaim 1, which further comprises a ribosome.
 18. The antibody-nucleicacid complex according to claim 1, wherein one molecule of the antibody,and one molecule of the nucleic acid encoding the target component arecontained per one molecule of the antibody-nucleic acid complex.
 19. Theantibody-nucleic acid complex according to claim 1, wherein the antibodyconsists of one H chain or an antigen binding portion thereof and one Lchain or an antigen binding portion thereof.
 20. The antibody-nucleicacid complex according to claim 1, wherein the antibody is a Fabfragment.
 21. The antibody-nucleic acid complex according to claim 1,wherein the nucleic acid is an mRNA or cDNA encoding any one targetcomponent of the antibody.